KRUMP LEA (IE)
| WO1985001441A1 | 1985-04-11 |
| FR2913338A1 | 2008-09-12 | |||
| US20070160683A1 | 2007-07-12 |
MARIA TORSEIN ET AL: "Risk factors for calf mortality in large Swedish dairy herds", PREVENTIVE VETERINARY MEDICINE, vol. 99, no. 2-4, 1 May 2011 (2011-05-01), NL, pages 136 - 147, XP055379970, ISSN: 0167-5877, DOI: 10.1016/j.prevetmed.2010.12.001
| CLAIMS: 1. A pharmaceutical or veterinary composition comprising a strong ion difference in the range of about 150-370 mmol/l, optionally about 165-370 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 2. The composition of Claim 1 comprising a strong ion difference in the range of about 150-270 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 3. The composition of Claim 1 comprising a strong ion difference in the range of about 270-370 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 4. The composition of Claim 1 comprising a strong ion difference in the range of about 200-370 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 5. The composition of Claim 2 comprising a strong ion difference in the range of about 200-270 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 6. A pharmaceutical or veterinary composition comprising, when dissolved in a diluent, optionally water, an alkalising agent, optionally bicarbonate, in the range of about 130 to 370 mmol/l, optionally about 130 to 300 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 7. The composition of Claim 6 comprising an alkalising agent, optionally bicarbonate, in the range of about 150 to 370 mmol/l, optionally about 130 to 300 mmol/l, when dissolved in a diluent, optionally water, further optionally water having a strong ion difference of less than about 10 mmol/l. 8. The composition of Claim 6 or 7, comprising, when dissolved in the diluent, a strong ion difference in the range of about 150-270 mmol/l. 9. The composition of any one of Claims 1 to 5, comprising, when dissolved in the diluent, an alkalising agent, optionally bicarbonate, in the range of about 130 to 370 mmol/l, optionally about 130 to 300 mmol/l. 10. The composition of any one of Claims 1 to 5, comprising, when dissolved in the diluent, an alkalising agent, optionally bicarbonate, in the range of about 150 to 370 mmol/l, optionally about 150 to 300 mmol/l. 11. The composition of any one of Claims 1 to 5, 9 and 10 comprising, when dissolved in the diluent, an alkalising agent, optionally bicarbonate, in the range of about 300 to 370 mmol/l. 12. The composition of any one of Claims 1 to 5, 9 and 10 comprising, when dissolved in the diluent, an alkalising agent, optionally bicarbonate, in the range of about 150 to 370 mmol/l. 13. The composition of Claim 6 or 7, comprising, when dissolved in the diluent, a strong ion difference in the range of about 150-370 mmol/l, optionally a strong ion difference in the range of about 200-350 mmol/l, further optionally a strong ion difference in the range of about 165-370 mmol/l, optionally a strong ion difference in the range of about 270-370 mmol/l, optionally a strong ion difference in the range of about 150-270 mmol/l. 14. The composition of any one of Claims 1 to 13, in which the sodium ion concentration, when dissolved in the diluent, is in the range about 190 to 390 mmol/l, optionally about 250 to 375 mmol/l, further optionally about 340 mmol/l. 15. The composition of any one of Claims 1 to 13, in which the sodium ion concentration, when dissolved in the diluent, is in the range about 250 to 480 mmol/l, optionally about 190 to 480 mmol/l, further optionally about 250 to 430 mmol/l, still further optionally about 340 mmol/l. 16. The composition of any one of Claims 1 to 15, in which the potassium ion concentration, when dissolved in the diluent, is in the range about 20 to 40, optionally about 27 mmol/l. 17. The composition of any one of Claims 1 to 16, in which the chloride ion concentration, when dissolved in the diluent, is in the range about 60 to 150 mmol/l, optionally about 75 to 145 mmol/l, further optionally about 130 mmol/l. 18. The composition of any one of Claims 1 to 16, in which the chloride ion concentration, when dissolved in the diluent, is in the range about 60 to 150 mmol/l, optionally about 75 to 145 mmol/l, further optionally about 115 to 145 mmol/l, still further optionally about 130 mmol/l. 19. The composition of any one of Claims 6 to 8 and 13, in which the alkalising agent, when dissolved in the diluent, is in the range of about 175 to 300 mmol/l, optionally about 200 to 300 mmol/l, further optionally about 237 mmol/l. 20. The composition of any one of Claims 6 to 8, 13 and 19, in which the alkalising agent, when dissolved in the diluent, is in the range of about 150 to 370 mmol/l, optionally about 200 to 370 mmol/l, further optionally about 237 mmol/l. 21. The composition of any one of Claims 6 to 20, in which the alkalising agent, when dissolved in the diluent, is selected from bicarbonate (HC03) or a bicarbonate precursor optionally selected from propionate, acetate or citrate; in which, optionally, in which the alkalising agent is bicarbonate (HC03). 22. The composition of Claim 21 , in which the alkalising agent is bicarbonate (HC03) that, when dissolved in the diluent, is in the range of about 175 to 300 mmol/l, optionally about 200 to 300 mmol/l, further optionally about 237 mmol/l. 23. The composition of Claim 21 , in which the alkalising agent is bicarbonate (HC03) that, when dissolved in the diluent, is in the range of about 150 to 370 mmol/l, optionally about 200 to 370 mmol/l, further optionally about 237 mmol/l. 24. The composition of any one of Claims 1 to 23, the composition comprising an osmolality in the range of about 750 to 1000 mOsm/l, optionally about 900 to 1000 mOsm/l, further optionally about 939 mOsm/l. 25. The composition of any one of Claims 1 to 23, the composition comprising an osmolality in the range of about 750 to 1300 mOsm/l, optionally about 900 to 1300 mOsm/l, further optionally about 939 mOsm/l. 26. The composition of any one of Claims 1 to 5, 9 to 12, 14 to 18 and 21 to 25, in which the strong ion difference, when dissolved in the diluent, is in the range of about 170 to 270 mmol/l, optionally about 215 to 255 mmol/l, further optionally about 237 mmol/l. 27. The composition of any one of Claims 1 to 5, 9 to 12, 14 to 18 and 21 to 2612, in which the strong ion difference, when dissolved in the diluent, is in the range of about 150 to 370 mmol/l, optionally about 200 to 370 mmol/l, further optionally about 215 to 300 mmol/l, still further optionally about.237 mmol/l. 28. The composition of any one of Claims 1 to 27, further comprising a water soluble vitamin, optionally, tocopherol. 29. A method of treating diarrhoea in a subject, optionally a domesticated animal, the method comprising administering the composition of any one of Claims 1 to 28. 30. The method of Claim 29, in which the subject is a domesticated animal that is optionally selected from calves, lambs, kids, foals, piglets, dogs, cats, further optionally is calves. 31. The method of Claim 29 or 30, in which the composition is administered in a one dose treatment protocol or, alternatively, in a two dose treatment protocol administered about 12 hours apart. 32. The method of Claim 31 , in which the, or each, dose is provided in a volume in the range of 0.25 to 5 litres. 33. The method of Claim 31 , in which the subject is a calf and the, or each, dose is provided in a volume in the range of about 1 to 3.5 litres, optionally about 2 litres. 34. The method of any one of Claims 29 to 33, in which the composition is administered without either milk or milk replacer. 35. The method of any one of Claims 29 to 34, in which the composition is administered per os. 36. The pharmaceutical or veterinary composition of any one of Claims 1 to 28 dissolved in about 0.25 to 5 litres of diluent. 37. The pharmaceutical or veterinary composition of any one of Claims 1 to 28 dissolved in about 1 to 3.5 litres, optionally about 2 litres of diluent. 38. The pharmaceutical or veterinary composition of any one of Claims 1 to 28, 37 or 38, comprising a strong ion difference of about 237 mmol/l, when dissolved in water, optionally water having a strong ion difference of less than about 10 mmol/l. 39. The pharmaceutical or veterinary composition of Claim 38, comprising, when dissolved in water, optionally water having a strong ion difference of less than about 10 mmol/l: 40. The pharmaceutical or veterinary composition of Claim 38 or 39, further comprising about 237 mmol/L of Bicarbonate. 41. The pharmaceutical or veterinary composition of any one of Claims 38 to 40, further comprising about 205 mmol/L of Dextrose. 42. The pharmaceutical or veterinary composition of any one of Claims 38 to 41 , having an osmolality of about 939 mmol/L. 43. The pharmaceutical or veterinary composition of any one of Claims 1 to 28 or 37 to 42, consisting of, when dissolved in water, optionally water having a strong ion difference of less than about 10 mmol/l: Na K mmol/L CI" mmol/L Dextrose Bicarbonate SID Osmolality mmol/L mmol/L (mmol/L) mmol/L mmol/L 340 27 130 j 205 237 237 939 44. A composition of any one of Claims 1 to 28 or 36 to 43 for use in a method of treating diarrhoea in a subject, optionally a domesticated animal, the use comprising administering the composition of any one of Claims 1 to 28 or 36 to 43. 45. The composition for use of Claim 44 in which the subject is a domesticated animal that is optionally selected from calves, lambs, kids, foals, piglets, dogs, cats, further optionally is calves. 46. The composition for use of Claim 44 or 45, in which the composition is administered in a one dose treatment protocol or, alternatively, in a two dose treatment protocol administered about 12 hours apart. 47. The composition for use of Claim 44 or 45, in which the, or each, dose is provided in a volume in the range of about 0.25 to 5 litres. 48. The composition for use of Claim 45, in which the subject is a calf and the, or each, dose is provided in a volume in the range of about 1 to 3.5 litres, optionally about 2 litres. 49. The composition for use of any one of Claims 44 to 48, in which the composition is administered without either milk or milk replacer. 50. The composition for use of any one of Claims 44 to 49, in which the composition is administered per os. |
administration protocols (Smith and Berchtold, 2014). This makes it difficult for producers, and veterinarians, to identify a product that best suits the needs of diarrhoeic animals, including calves. European directive, 2008//38 EC (amendment M4, 22 October 2014), sets requirements and recommendations for an ORBS to be suitable for treatment of electrolyte imbalance and acidosis in calves. It emphasises a minimum SID value of 60 mM for such therapies. Based on their recommendations, the SID can range from 65 to 138 mmol/l (see table below):
Based on the interpretation of Stewart (1981 ), SID is regarded as the major factor in determining the alkalising abilityof an ORBS and as a valid approach when formulating an ORBS for calves with diarrhoea and metabolic derangement (Stampfli et al., 2012). The question as to which is more important, an ORBS with high SID, or an ORBS with an alkalinising agent has yet to be definitively answered (Smith and Berchtold, 2014). There is also no consensus on a suitable alkalinising agent. The use of bicarbonate precursors such as acetate or propionate are favoured over bicarbonate for their energy value once metabolised, their water absorption capabilities and that they do not alkalinise the abomasum. Bicarbonate was believed to inhibit abomasal milk clotting, however this has not been supported by more recent studies (Bachmann et al., 2009;
Constable et al., 2009). Unlike medicines, which undergo rigorous testing prior to European Commission approval, ORBSs within Europe are not assessed for clinical efficacy before market placement. While a limited number of studies have assessed various aspects of ORBS treatment in natural neonatal calf diarrhoea (Constable et al., 2009; Grunberg et al., 2013; Kirchner et al., 2014; Naylor, 1989; Stampfli et al., 2012; Stampfli et al., 1996), there is a lack of observational field studies in recent years examining the efficacy and suitability of ORBSs for use in calf diarrhoea (Meganck et al., 2014), in particular data relating to an ORBS conforming to recently amended European Union (EU) legislation. In an attempt to increase scientific knowledge in this area, the aim of this observational study was to investigate outbreaks of calf diarrhoea on several dairy farms using rapid 'pen-side' blood gas analysis and subsequently to evaluate treatment of diarrhoeic calves using an ORBS that is compliant with current EU legislation. Neonatal calf diarrhoea is an infectious disease of bacterial, virial or protozoal origin that is invariably followed by dehydration and metabolic acidosis. The treatment for calf diarrhoea (regardless of the cause) is based on fluid therapy, most commonly using oral rehydration solutions (ORS). These solutions usually contain the basic ingredients of glucose, sodium (Na+), potassium (K+) and chloride (CI-). Often a buffer, such as bicarbonate or its precursor, is added to address the acidosis. The scientific literature has recently focused on a strong ion difference (SID) approach to determine the suitability for purpose of a rehydration solution. SID is used as a proxy for buffering capacity (Stewart, 1981 ). The term "SID", as used herein, is calculated using the concentration of three ions in the solution with the following formula: [Na+] + [K+] - [CI-]. The current scientific recommendations for SID are between 60 mmol/l (Smith and Berchtold, 2014) and 1 10 mmol/l (Stampfli et al., 2012) although an optimal SID for a solution has not yet been determined. Our research, both in vitro and in vivo, investigated alternative scientific approaches to using an oral rehydration solution. Our aim was to re-evaluate the conventional scientific knowledge on the best and most appropriate solution for the treatment of calves with diarrhoea and acidosis. 76
77 Our invention goes directly against the conventional scientific wisdom on the most suitable SID for a
78 rehydration solution. Most of the ingredients in our product are similar to other products but their
79 concentration and combination differs significantly. Our formula is different in the following ways: 80
81 The SID of our formula is innovative in its concentration. We took the 'non-obvious' approach by
82 investigating concentrations that went well beyond the scientific recommendations - from 150 and up
83 to 370 mmol/l. The concentration we now claim goes against the conventional wisdom and, to the
84 best of our knowledge, our closest competitor has an SID concentration of 140 mmol/l - taken from
85 Smith and Berchtold, 2014. We currently have the highest SID ever to be used in a product for calf
86 diarrhoea which accounts for the efficacy.
87
88 The SID of the formula is our main focus, since it is the key to improved efficacy of our product. An
89 investigation of the key electrolyte manufacturers (Appendix 1 below) indicates SID values all well
90 below 100, matching the current recommendations.
91
92 To the best of our knowledge, the highest SID value of a solution ever used for research purposes
93 was 189 mmol/l (Bachmann et al., 2009). However, to achieve this value, they had used double the
94 concentration of the oral rehydration solution than was recommended by the manufacturer and the
95 solution was prepared with milk replacer, which has its own SID of 42 mmol/l and osmolality of 287
96 mOsm/kg. Therefore, the solution prepared in water (even by doubling the concentration) would have
97 an SID of 147 mmol/l and an osmolality of 633 mOsm/kg.
98
99 The research article by Bachmann et al. (2009) reflects ad hoc research investigating the potential
100 negative impact of SID (of a solution) on abomasal emptying. The authors attempted to purposely
101 stray outside the scientifically accepted norms of SID ranges and investigate extreme values to draw
102 reasonable conclusions on this effect. They did not investigate diarrhoea but rather focused on
103 healthy calves for a different purpose, whereas our invention was assessed in sick calves with the
104 sole purpose of treating diarrhoea in those calves.
105
106 It is evident that "only plasma volumes of groups fed MR [milk replacer] and MR-ORS [milk replacer -
107 oral rehydration solution] mixtures were still increased, whereas plasma volumes of groups fed water-
108 ORS mixtures were back to baseline. The MR-ORS mixtures were most effective in increasing
109 plasma volume at the 2 determined time points, reaching statistical significance compared with MR or
1 10 water-ORS mixtures 240 min after feeding (P < 0.05). " (Bachmann et al. , 2009).
1 1 1
1 12 Therefore, Bachmann et al. (2009) have concluded that "administration of these MR-ORS mixtures
1 13 causes a more pronounced expansion of plasma volume, which is beneficial for the correction of
1 14 dehydration in diarrheic calves. "
1 15 In contrast to that, we have found an optional formula that has an SID of 237 mmol/l by mixing it with water alone, which has been proven to be beneficial for the correction of dehydration in calves with diarrhoea. Indeed, subsequent analysis on this topic by the same group (Bachmann et al., 2012) focuses the research at an SID range of 35 - 84 mmol/L, further advancing the ad hoc nature of the 2009 investigation with an SID of 189 mmol/L which was employed to test the extremes on abomasal emptying alone. Additionally, Bachmann et al did not choose to advance studies on the high SID values but on another electrolyte (Lytafit) because "it was shown that the preparation of Lytafit in MR did not impair abomasal milk clotting". From this study, Bachmann et al. (2012) generates two conclusions on SID - (a) "an ORS with a
[SID3] of 84 mmol/L increased plasma [SID3] in healthy calves, indicating that, for effective correction of metabolic acidosis in diarrheic calves, ORS should contain high [SID3] values", and (b) "an ORS with [SID3] values >80 mmol/L, which expand [SID] in healthy calves, should be used in oral rehydration treatment of diarrheic calves". It is proposed that Bachmann's SID focus was in the range of 84 mmol/L and the use of the term 'high' ("ORS should contain high [SID3] values') was presented/stated in the context of the prevailing theory that the most favourable SID was 60 - 80 mmol/L (Smith and Berchtold, 2014). The use of milk or water as an ORS diluent has been the subject of much research. From their own studies, Bachmann et al. (2009 & 2012) conclude that milk (or milk replacer) is the preferred option. It is stated by these authors that "only plasma volumes of groups fed MR [milk replacer] and MR-ORS [milk replacer - oral rehydration solution] mixtures were still increased, whereas plasma volumes of groups fed water-ORS mixtures were back to baseline. The MR-ORS mixtures were most effective in increasing plasma volume at the 2 determined time points, reaching statistical significance compared with MR or water-ORS mixtures 240 min after feeding (P < 0.05). " (Bachmann et al . , 2009).
In addition, the MR-ORS solution with an SID of 189 mmol/l contained acetate as the alkalising agent, which Smith and Berchtold, 2014 has demonstrated has several advantages over bicarbonate, whereas our invention optionally contains bicarbonate and has, surprisingly, been proven to be beneficial for the correction of acidosis in calf diarrhoea.
Alternatively, or additionally, the osmolality of our formula is 750 to 1300, optionally, 750 to 1000 or 900 to 1000, further optionally, about 939 mOsm/l - this is significantly higher than that of an average ORS. According to our research, the product with the highest osmolality is Entrolyte HE (Zoetis, USA) with an osmolality of 739 mOsm/l (taken from Smith and Berchtold, 2014). However, current scientific recommendations advise against ORS with an osmolality higher than 750 mOsm/l (Nouri and Constable, 2006). 155 Alternatively, or additionally, the present composition comprises a significantly increased content of
156 an alkalising agent in the range, when dissolved in a diluent, in the range of 150 - 370 or 130 - 300
157 or 130 - 370 mmol/l, for example, we used Sodium Bicarbonate as an alkalising agent at a
158 concentration of about 237 mmol/l. The current scientific recommendation for an ORS is to contain
159 50-80 mmol/l of an alkalising agent (Smith, 2009) and most products on the market contain 80 mmol/l
160 or less of an alkalising agent (Appendix 1 ).
161
162 The interplay between alkalising agent and SID in acid-base assessment has been the subject of
163 significant research - as reviewed by Constable et al., 2014. The traditional approach for assessing
164 acid-base balance in animals uses the Henderson-Hasselbalch equations and focuses on how
165 plasma pH is determined by the interaction between carbon dioxide tension (PC0 2 ), the bicarbonate
166 concentration (cHC0 3 _), the negative logarithm of the apparent dissociation constant (pK10) for
167 carbonic acid (H 2 C0 3 ), and the solubility coefficient for C0 2 in plasma (S). This equation, however, is
168 limited to healthy animals with plasma protein concentrations within the reference range, and cannot
169 be applied to neonatal calf diarrhoea. New models based on strong ions, developed by Stewart
170 (1981 ) and refined by Constable (2000), established a mechanistic acid-base theory that can provide
171 an enhanced understanding of acid-base disturbances in pathological cases. This theory indicates
172 that it is the sodium in sodium bicarbonate and, therefore, the SID that is important in correcting the
173 acid-base disturbance in a diarrhoea calf. Applying the Henderson-Hasselbalch equation to the same
174 situation, however, suggests that it is the bicarbonate in sodium bicarbonate that is important
175 (Constable et al., 2014). Differentiating both theories experimentally has not been achieved to date,
176 as both theories are inextricably linked. .
177
178 The present invention, in relation to SID and, separately, alkalising agent has greatly enhanced the
179 efficacy of our formula relative to existing formulations. Our formula has proven to be effective with
180 proven recovery as soon as 6 hours, representing an unprecedented improvement in recovery time,
181 relative to the current art. Figure 1 & Figure 5 below demonstrate proof of this decreased time to
182 recovery, thereby improving the animal's health and welfare and subsequently decreasing mortality. 183
184 The composition of the present invention is also effective with a two-dose treatment protocol. This is
185 significantly less than competitor products (Appendix 2). This two-dose treatment protocol increases
186 compliance by the end user, making it more time efficient.
187
188 The composition of the present invention has been tested against extreme cases, as determined on
189 the basis of pH. In the study, animals with pH values as low as 6.7 were recorded (amongst the
190 lowest values recorded in the literature), and made a full recovery with the standard treatment.
191
192 Calves treated with the composition of the present invention have a growth rate comparable to control
193 (healthy) calves, in spite of the effects of disease (see, for example accompanying Figure 4). The 194 composition of the present invention enables better animal productivity and is therefore of a major
195 economic benefit to the producer (farmer).
196
197 Optionally, the composition of the present invention may comprise tocopherol (vitamin E). Based on
198 our knowledge and research, this vitamin has never been used for treatment of calf diarrhoea. Since
199 vitamin E is a well-recognised antioxidant and plays an important role in calf health (Torsein et al.,
200 201 1 ), it is may play a role in the fast recovery using the composition of the present invention.
201
202 The composition of the present invention, while a treatment for scour in itself and exemplified
203 hereunder in relation thereto, also has the potential for further medicinal applications where diarrhoea
204 is also an outcome. This can be achieved by the addition of either medical (e.g. antibiotics or
205 coccidiostats for bacterial or parasitic disease, respectively) which will further improve the efficacy this
206 targeted market, or other ingredients (e.g. to improve palatability).
207
208 While the composition of the present invention is exemplified hereunder in relation to the treatment of
209 calves, the composition of the present invention is also applicable to the treatment of diarrhoea in
210 other domesticated animal species (lambs, kids, foals, piglets, dogs, cats, etc.), as well as, in
21 1 humans.
212
213 By "domesticated animal" is meant animals, including animals which are or may be undergoing the
214 process of domestication and animals that have an extensive relationship with humans beyond simple
215 predation. This includes species which are semi-domesticated, undomesticated but captive-bred on a
216 commercial scale, or commonly wild-caught, at least occasionally captive-bred, and tameable.
217 Archaeozoology has identified 3 classes of animal domesticates: (1 ) commensals, adapted to a
218 human niche (e.g., dogs, cats, guinea pigs); (2) prey animals sought for food (e.g., cows, sheep, pig,
219 goats); and (3) targeted animals for draft and non-food resources (e.g., horse, camel, donkey), all of
220 which are include in "domesticated animal".
221
222 By "calf" is meant a bovine animal under the age of 6 months.
223
224 By "bicarbonate" is meant the anion - HC0 3 " . By "SID", is meant the concentration of three ions in the
225 solution with the following formula:
226
227 [Na+] + [K+] - [CI-] = [SID]
228
229 In the drawings,
230
231 Figure 1. Clinical assessment scores (CAS) for diarrheic calves, pre- and post-ORBS treatment. As
232 used herein, "ORBS" is "Vitalife", defined hereunder.
233 Pre-ORBS: prior to administration of ORBS 234 Post (6-18): 6 to 18 hours post-administration of ORBS
235 Post (24-48): 24 to 48 hours post-administration of ORBS
236 * Percentage of diarrheic case calves
237
238 Figure 2. Mean blood pH, HC0 3 std , base excess blood (BEB), and anion gap (AG) (± SEM) at each
239 clinical assessment score* (CAS).
240 *CAS groups 3 and 4 were merged for analysis. (CAS 0, n=18 (27 data points); CAS 1 , n=2Q (27 data
241 points); CAS 2, n=10 (13 data points); CAS 3, n=7 (18 data points), CAS 4, n=1 (1 data point)). 242
243 Figure 3. Mean blood sodium (Na + ), potassium (K + ), Chloride (CI ) and strong ion difference (SID) (±
244 SEM) at each clinical assessment score* (CAS).
245 *CAS groups 3 and 4 were merged for analysis. (CAS 0, n=18 (27 data points); CAS 1 , n=2Q (27
246 data points); CAS 2, n=10 (13 data points); CAS 3, n=7 (18 data points), CAS 4, n=1 (1 data point)). 247
248 Figure 4. Weight measurement over time for healthy (n=24) and ORBS-treated diarrhoea (n=8)
249 calves.
250 Data records were available for farm A calf cohort only.
251 Arrow highlights commencement of diarrhea outbreak in this calf cohort.
252
253 Figure 5. Comparative assessment of pH, bicarbonate (HC0 3 ~ ), Base Excess, SID (blood SID of the
254 calf) and Anion Gap (AG) following administration of one of three [SID] solutions for the treatment of
255 neonatal calf diarrhoea. Pre-treatment values were standardised by subtracting this value from the
256 pre- and the post-treatment values. The horizontal dashed line on each graph is presented as the
257 normal value for each variable. The three solutions had an [SID] of 95 mmol/L (n=2), [SID] of 155
258 mmol/L (n= 1 ), and [SID] of 237 mmol/L (present invention) (n=2), respectively.
259
260 Appendix 1 below compares an optional embodiment of the present invention with other similar
261 products on the market in terms of concentration of Bicarbonate, Sodium, Chloride as well as SID and
262 osmolality.
263
264 265 Hereunder, the terms "Vitalife" or "Vitalife for Calves" are used interchangeably herein to refer to a
266 composition, when dissolved in water, that comprises:
267 Table A "Vitalife" or "Vitalife for Calves" - an optional embodiment of the present invention:
J Na K mmol/L CI ' mmol/L Dextrose Bicarbonate SID Osmolality mmol/L mmol/L (mmol/L) mmol/L mmol/L
340 27 130 205 237 237 939 I
268
269 Tables below (both identified as Appendix 2) compare the dose rate and time to recovery of several
270 different products on the market.
271
272 Appendix 2 : Number of sachets per calf
276 277
278 INDEPENDENT STUDY 1
279
280 MATERIALS AND METHODS
281
282 An observational study was conducted on dairy calves (51 healthy, 31 calves with neonatal diarrhea)
283 during outbreaks of diarrhoea on four dairy farms. Clinical assessment scores (CAS) were assigned
284 to each healthy and diarrhoeic calf (0 (healthy) to 4 (marked illness)). Blood gas analysis (pH, base
285 excess (BE), Na+, K+, Ca2+, CI-, glucose, total haemoglobin, standard HC0 3 " , strong ion difference
286 (SID), and anion gap (AG)) was completed for each calf. Repeated measurements were taken in
287 healthy animals, and pre- and post-intervention measurements taken for diarrheic calves. The mean
288 CAS of diarrheic calves was 1.7, with 51 %, 30%, 17% and 2% of calves scoring 1 , 2, 3 and 4,
289 respectively. The mean values for blood pH, BE, AG and SID was 7.26, -4.93mM, 16.3mM and
290 38.59mM, respectively. Calves were administered an oral rehydration and buffering solution (ORBS;
291 "Vitalife" - see Table A) and reassessed. The mean CAS at 6 to 18 hours post-treatment was 0.38
292 (65% of calves scored 0 and 35% scored 1 ), which reduced to 0.03 (98% of calves scored 0 and 2%
293 scored 1 ) within 24 to 48 hours. A significant increase in mean values (P<0.001 ) for pH, BE, HCO 3 -,
294 Na+ and SID was recorded post-treatment and a significant decrease in AG, K + , Ca 2+ and total
295 haemoglobin. The correlation estimates indicated that pH, HC0 3 " and BE were strongly correlated
296 with CAS, with values exceeding 0.60 in all cases (P<0.05). Administration of "Vitalife" (see Table A),
297 an ORBS with a high SID and bicarbonate buffer, demonstrated rapid recovery from a diarrhoeic
298 episode in dairy calves.
299
300 Clinical Assessment Score (CAS)
301
302 In order to comparatively assess diarrhoeic calves pre- and post-treatment, a five point clinical
303 assessment scoring chart (CAS) was used. The chart was developed for use by farm managers and
304 veterinarians at Teagasc (Irish Agriculture and Food Development Authority, Ireland) dairy research
305 farms. Clinically healthy calves were assigned a CAS of 0, with varying degrees of ill-health scored in
306 increments of 1 , to a maximum of 4. We constructed the chart based on previously published
307 dehydration charts (Naylor, 1989) and the Wisconsin respiratory calf health scoring model (McGurik,
308 2008). The chart incorporated calf demeanour, ear position, mobility, suckle reflex, enophthalmos
309 and desire-to-feed variables. Temperature was not recorded, as the study sought to use variables
310 most indicative of dehydration and metabolic acidosis, and variables which would also be routinely
31 1 observed by producers on commercial farms. Additionally, no attempt was made to identify the
312 underlying cause of the diarrhoea as it was not the focus of the research. Clinical assessment was
313 completed prior to each blood sample taken, and in the case of diarrhoeic calves, an additional
314 assessment was conducted at 24 to 48 hours post-treatment. All calves were assessed and scored
315 simultaneously by two research veterinarians and a single consensus score recorded. All CASs were
316 recorded prior to generation of blood gas results. 317
318 Sample Population
319
320 An observational study of 77 calves from two research (A and B) and two commercial (C and D) dairy
321 farms was completed over a 21-day period in spring 2015. A description of husbandry regimes on
322 each study farm for calves in the first month of life is presented in Table 1. Calves were defined as
323 clinically healthy if they recorded a CAS of 0 (as previously described) and had no evidence of
324 diarrhoea. Healthy calves were sampled on farm A during a period when no cases of diarrhoea had
325 been recorded on the farm from the start of the calving season to the time of assessment (n=28, 71
326 measurements). Healthy case animals were also identified on farms B (n=4, 6 measurements) and C
327 (n=19, 19 measurements) during a period of diarrhoea outbreak on those farms. Diarrhoeic case
328 calves were defined as having a CAS of 1 or greater, and evidence of diarrhoea. Such calves were
329 identified on farms B (n=9), C (n=2) and D (n=12). A diarrhoea outbreak subsequently occurred on
330 farm A which facilitated analysis of an additional 8 calves, five of which were sampled earlier as part
331 of the healthy cohort. All animals, both healthy and diarrhoeic, were enrolled to the study between the
332 ages of 7 and 26 days.
333
334 Sampling and Administration of ORBS - "Vitalife" - see Table A
335
336 Each case calf was blood sampled by jugular venipuncture on at least one, but not more than three,
337 occasions over the duration of the study. Venous blood samples were taken into heparinized 1 mL
338 syringes (Cruinn Diagnostics, Dublin, Ireland), immediately placed on a bottle roller, and continuously
339 agitated for at least 20 seconds to avoid formation of microdots. Prior to testing, all visible air bubbles
340 were expelled from the syringe. A bench top Rapidpoint 400 (Siemens, Munich, Germany) analyzer
341 was used to test all samples. Parameters reported by the analyzer included pH, base excess (BE;
342 mM), Na + (mM), K + (mM), Ca 2+ (mM), CI " (mM), Glucose (mM), total haemoglobin (Hb; g/dL), standard
343 HC0 3 " (mM), and anion gap (AG; mM). For healthy calves, samples were taken over a period of three
344 days, approximately two hours post-feeding. In the case of diarrhoeic calves, pre-treatment samples
345 were taken within two hours of a milk feed being offered (many of the diarrhoeic calves had
346 diminished suck reflexes and either fed to a limited degree or not at all). These calves were then
347 administered an ORBS ("Vitalife" - see Table A) reconstituted in water according to manufacturer's
348 instructions. All treatments were administered by oesophageal tube. Post-treatment blood samples
349 were collected between six and 18 hours following ORBS intervention.
350
351 Additional Calf Data
352
353 Accurate calf date of birth, sex, breed, birth weight, whether the calf was a singleton or twin, and the
354 level of calving difficulty experienced by the dam were available for all calves from farm A. On-going
355 regular weight data (weekly or biweekly) were only available from farm A, and included measurement
356 on all 8 diarrhoeic calves, and 20 of the 23 healthy calves. 357
358 Data Analysis
359
360 Data management and graphical representations were completed using Microsoft Excel (Microsoft
361 Office 2010, Microsoft Corporation, Redmond, Washington, USA).
362
363 Preliminary steps established the stability of the variance for each of the continuous variables. For
364 the purposes of analysis, results for calves recording CASs of 3 and 4 were grouped. Associations
365 between various genetic and environmental factors and blood gas measurements were investigated
366 in the healthy calf cohort from Farm A. A cross-sectional, time series, generalized estimating
367 equation (Stata: xtgee procedure) was used to account for repeated measures. The model tested the
368 combined effect of sex, calving difficulty, breed, date of calving, birth weight and single or twin on
369 each of the 1 1 blood gas variables (pH, HC0 3 " , BE, AG, Na + , K + , Cl + , Ca 2+ , Glucose, total
370 haemoglobin and SID). Each model was constructed using a Gaussian distribution, identity link and
371 an exchangeable correlation. The effect of ORBS treatment on the 1 1 blood gas measurements of
372 diarrhoeic calves was assessed by linear regression, with status (pre- and post-ORBS treatment)
373 used as the independent variable. The effect of environment (diarrhoea or diarrhoea-free) on the 1 1
374 blood gas measurements of healthy calves was assessed by linear regression, with environmental
375 status used as the independent variable. A further linear regression model was constructed to assess
376 the effect of ORBS treatment on CAS. A student t-test was used to comparatively assess calf weight
377 at the at weekly or biweekly weight measurement time points. A Spearman correlation was used to
378 determine the association between each blood gas variable and CAS. To achieve this, the
379 continuous blood gas variables were reclassified as ordinal data using the standard deviation value
380 for each variable as an increment gap size. Each increment was ranked sequentially on an ordinal
381 scale, with 1 classed as the lowest in each case. Finally, a Pearson correlation was used to
382 determine the association between bicarbonate and SID. P values of <0.05 were considered
383 statistically significant. All statistical analysis was performed using Stata/SE v12.1 (StataCorp, Texas,
384 USA).
385
386 Study Approval
387
388 This study was approved by the Teagasc Animal Ethics Committee (TAEC 81/2014), all procedures
389 were authorized and carried out in accordance with the Health Products Regulatory Authority (HPRA)
390 of Ireland (AE19132/P037).
391
392 393 RESULTS
394
395 The blood gas profile of diarrhoeic calves is presented in Table 2, with healthy calf values presented
396 for comparative purposes. The treatment of diarrhoeic calves with "Vitalife" (see Table A), an EC-
397 compliant ORBS, led to a significant increase in mean values (P<0.001 ) for pH, BE, HC0 3 " , Na + and
398 SID relative to pre-treated diarrhoeic calf values, while a significant decrease was recorded for AG,
399 K + , Ca 2+ and total Hb. None of the 31 "Vitalife'-treated animals died during the post-monitoring
400 clinical assessment period of 8 days. On the research farms A and B where longer term records were
401 maintained, all treated animals made a full recovery, as determined by CAS values of 0 and were
402 returned from hospital facilities to the general calf population.
403
404 The blood gas results of healthy calves reared in a diarrhoea-free, and in a diarrhoea environment are
405 presented in Table 3. With four exceptions (bicarbonate, SID, BE and AG), these results correspond
406 with previously published reference ranges. Statistical comparisons between these two groups
407 indicated that the calves reared in a diarrhoea environment had significantly lower values for pH, AG,
408 Na + , CI " and glucose.
409
410 The CAS for pre- and post-ORBS (specifically "Vitalife" - see Table A) treated diarrhoeic case calves
41 1 are presented in Figure 1. The mean CAS for pre-treatment diarrhoeic calves was 1.7, with 49% of
412 cases recording a CAS of 2 or more. Following ORBS treatment, the average CAS was reduced to
413 0.38, with 65% of cases recording a CAS of 0 (clinically healthy) indicating a generalized shift
414 amongst all treated animals towards a healthy clinical status. Within 48 hours of ORBS treatment, all
415 animals with a single exception, had a CAS value of 0 (mean CAS of 0.03).
416
417 The correlations between CAS and blood gas variables are presented in Table 4. The correlation
418 estimates indicate that pH, HC0 3 " , and BE were strongly and significantly correlated with CAS, with
419 values exceeding 0.60 in all cases (P<0.05). A further correlation analysis between bicarbonate
420 concentration and SID yielded a correlation estimate of 0.78 (P<0.0001 ). Graphical representations
421 of 8 blood gas variables and CAS are presented in Figures 2 and 3.
422
423 Weight measurements, recorded from research farm A, in healthy and ORBS treated diarrhoeic
424 calves, are presented in Figure 4 and no significant difference in weights was identified at any time
425 point (P>0.05 in all cases). Prior to the outbreak, the diarrhoea cohort had a non-significant heavier
426 mean weight relative to the healthy cohort. In a 14-day period following the diarrhoea outbreak, this
427 weight advantage was temporarily reversed indicating reduced growth rates in the diarrhoea cohort.
428 However, similar mean weights were recorded thereafter for both cohorts.
429
430 The assessment of the effect of sex, calving difficulty, breed, date of calving, weight and single/twin
431 on blood gas variables at birth indicated no significant associations.
432 433 DISCUSSION
434
435 Blood gas analysis was used in this observational study to assess both healthy, and pre- and post- 436 ORBS (specifically "Vitalife" - see Table A) treated diarrhoeic dairy calves. We found blood pH to be
437 a simple and useful indicator of clinical health in study calves and would be a useful diagnostic and
438 prognostic tool at farm level. Additionally, we observed that "Vitalife" (Table A), an ORBS which
439 couples a high SID and a bicarbonate buffer, is an appropriate treatment for diarrhoeic calves. It
440 effectively restored blood gas parameters to concentrations comparable to healthy animals, and all
441 animals treated in the study recovered rapidly from the diarrhoeic episode.
442
443 The strong significant correlation we identified between CAS and pH, bicarbonate, and BE, in
444 particular, indicates that clinical health is determined more by bicarbonate concentration than any of
445 the electrolytes measured. This is in agreement with a number of previous studies (Geishauser and
446 Thijnker, 1997; Kasari and Naylor, 1984; Lorenz, 2004; Naylor, 1989; Wendel et al., 2001 ), where the
447 link has been well established. Typically, a diarrhoeic calf will be hyponatremic, and hypo- or hyper-
448 kalemic (Constable and Griinberg, 2013; Lewis and Phillips, 1973) based on the chronic or acute
449 stage of the condition (Smith and Berchtold, 2014), respectively. However, the pre-treatment
450 diarrhoeic calves in this study had a wide range of electrolyte concentrations, with no clear consensus
451 as to a definitive hypo- or hyper- status for either sodium or potassium.
452
453 We would suggest, therefore, that for an individual diarrhoeic calf, assessment of sodium and/or
454 potassium electrolyte concentrations is an unreliable indicator of the severity of the diarrhoea.
455 However, the fact that bicarbonate was strongly associated with SID in these calves may support the
456 theory that it is the intra-relationship between these elements, in addition to chloride, that is a more
457 important associate to bicarbonate concentration rather than the individual elements themselves. 458
459 The CAS chart was used purely as a means of formalizing and standardizing assessment of calf
460 health over the duration of this study. It is not presented, nor is it intended, to act as a validated
461 scoring tool to inform the timing of intervention nor treatment of diarrhoeic calves. In this study,
462 however, we have highlighted the parameters, i.e. blood pH, bicarbonate, BE, AG, that should be
463 used in validating such a scoring system.
464
465 Assessment of healthy calves, both in a diarrhoea-free and a diarrhoeic environment, are broadly in
466 line with previously published reference ranges (Divers and Peek, 2007; Smith, 2015). However, it
467 should be noted that these reference values relate to adult bovine animals, as there is limited
468 availability and variable ranges (Slanina et al., 1992) in the literature, for neonates. We identified
469 possible exceptions to adult reference ranges published previously. For example, the lower pH
470 reference range value of 7.31 , if theoretically applied to a calf in the current study, would be
471 considered clinically unhealthy, with a CAS of 1. Reference ranges for bicarbonate, SID and base
472 excess were also underestimated relative to the healthy animals in this study, with AG overestimated. 473 The timing of blood analysis relative to feeding is a possible factor to the variations in these variables.
474 The animals in this study were measured approximately two hours post-feeding. Age of the calf can
475 also have a determining factor on acidaemia (Naylor, 1989), with calves during their first week of life
476 less acidaemic than older calves. While we did not account for age in this study due to lack of
477 accurate records on commercial study farms, the youngest diarrhoeic calf was 7 days old, thus
478 unlikely to be naturally less acidaemic. Additionally, the fact that healthy calves reared in a diarrhoea-
479 environment had significantly lower blood gas values, relative to those in a diarrhoea-free
480 environment, for five of the 1 1 variables investigated, the possibility of management/environmental
481 influences on blood gas parameters is raised. As further data relating to blood gas measurements for
482 healthy neonate calves emerge from future studies, taking feeding time, neonate age (Mohri et al.,
483 2007) and environment stressors into account, it is likely that currently reported reference ranges
484 need to be revised. Blood gas analysis can be valuable for establishing baseline parameters,
485 confirming a diagnosis, determining the prognosis, planning therapeutic options and monitoring
486 response to treatment (Russell and Roussel, 2007), despite overestimating oxygen exchange fraction
487 in some cases (Detry et al., 2003). The results of the current study support its usefulness in the field
488 by allowing detection of electrolyte disturbance and acidosis in calves, and informative monitoring of
489 calf recovery post-ORBS treatment. The high cost of blood gas analyzers and widespread use by
490 veterinarians of clinical assessment alone in assessing calf diarrhoea precludes the use of this
491 accurate diagnostic tool at farm level. The strong correlation between pH and clinical health, as
492 measured in this study, would suggest that monitoring pH alone is useful, particularly as a measure of
493 monitoring recovery following treatment. The availability of more simplified, economical and portable
494 diagnostic equipment, such as a pocket blood pH meter would therefore improve accurate diagnosis
495 and prognosis based on our results. Identifying a suitable pH cut-off value below which (further)
496 treatment is required, would need to be established. Bleul et al. (2007) suggests a pH cut off of 7.20
497 to classify newborn calves as acidotic, while the lower reference range for pH is 7.31 for older
498 animals. However, on the basis of this current analysis, a value closer to 7.36 (mean pH value for
499 calves with a CAS value of 1 ) may be more appropriate for calves aged seven to 26 days. Further
500 analysis on a larger dataset, would be of benefit in defining a suitable cut-off value.
501
502 We chose "Vitalife" (Table A) to reflect a new range of electrolyte treatments that meet with
503 specifications of the modified EC directive. In Ireland, at least, all ORBSs must conform to this
504 directive. The exact formulation of the ORBS used in the current study had not previously been
505 disclosed for commercial reasons. "Vitalife" can now be disclosed as being a water-based ORBS
506 differentiated by high SID that includes a bicarbonate buffer and additional ingredients including fat
507 soluble vitamins. High SID alone may be sufficient as the central component of an ORBS and, when
508 optionally combined with a buffering component, the objective of restoring calves to full health is
509 achieved. The beneficial properties of sodium bicarbonate based buffers in ORBSs has been
510 previously reported (Sen et al., 2009) and the results of the current study (reduction in CAS and
51 1 normalisation of blood gas parameters) would suggest that coupling a high SID ORBS with a buffering
512 component yields an effective diarrhoea treatment. However, the ORBS we used ("Vitalife") contains 513 additional supplements, such as tocopherol. Its contribution to the efficacy of the product cannot be
514 disregarded in light of the important role fat soluble vitamins play in maintaining calf health (Torsein et
515 al., 201 1 ).
516
517 CONCLUSION
518
519 Administration of "Vitalife" ® , an ORBS formulated on a principle of high SID, coupled to a bicarbonate
520 buffer and supplementary nutritional ingredients, demonstrated rapid recovery from a diarrheic
521 episode in dairy calves - the composition of Vitalife is set out in Table A. Additionally, we observed
522 measurement of blood pH to be a useful and practical tool in monitoring calf recovery following
523 treatment for diarrhoea.
524
525 INDEPENDENT STUDY 2
526
527 Objective
528
529 To determine the optimal SID concentration to effectively restore deranged blood gas parameters to
530 normal values, following a diarrhoea episode in calves.
531
532 Materials and methods
533
534 The study was completed on on a research dairy farm, where calves suffering from diarrhoea were
535 identified. Each calf (n=7) was clinically assessed and given a clinical assessment score as per
536 Independent Study 1. Additionally, each calf was blood sampled by jugular venepuncture for blood
537 gas analysis. Five of the seven calves were randomly allocated into one of three groups and given
538 one of three treatment solutions, each mixed in 2L of warm water and administered orally. The three
539 test solutions were:
540
541 · 95 mmol/L [SID] (upper range of current scientific recommendations);
542 · 155 mmol/L [SID] (the lower range of our SID claim range, and 15 mmol/L higher than the
543 closest competitor in terms of the [SID] of a treatment solution);
544 · 237 mmol/L [SID] the [SID] of our invention.
545 Calves were re-evaluated 6 hours post-treatment, when another blood sample was obtained for blood
546 gas analysis. For ease of comparison and standardisation of results, mean pre-treatment values for
547 each treatment were subtracted from pre- and post-treatment values. This approach placed all pre-
548 treatment values for each treatment at 0 and facilitated comparative change in the post-treatment
549 assessment. Using two animals per group, statistical power of 0.90 was achieved, given the
550 predicted low standard deviation in the mean difference in treatments.
551
552 553 Results
554
555 Of the seven calves initially assessed as having evidence of neonatal calf diarrhoea, five had a CAS
556 of 1 and mild derangement of blood gas valuables pre-treatment. One animal was given a CAS of 0,
557 with normal blood gas variables and another was given a CAS of 3 and had severe derangement of
558 blood gas variables. Subsequently, only the five animals, with comparable diarrhoea severity (blood
559 gas and clinical assessment) levels, were included in the treatment analysis.
560
561 The results are presented in Figure 5, and indicate that the conventional scientific recommendation of
562 an [SID] of 80-100 mmol/L is incapable of normalisation of blood gas parameters within the
563 assessment timeframe of six hours. While the mid-range [SID] of 155 mmol/L achieved a better
564 grade towards normalisation of the blood parameters, only Vitalife for Calves, with an [SID] of 237
565 mmol/L, achieves the degree of normalisation required to class a calf as healthy, both clinically and
566 objectively with blood gas analysis within the assessment timeframe of six hours.
567
568 Discussion
569 The five calves with diarrhoea in this study would be classed, at the point of analysis, as 'mild' cases,
570 with blood pH values not below 7.30 and moderately negative base excess values. On average, the
571 SID 95 group had the lowest degree of blood gas derangement (closest to healthy levels) and SIDvit a ii fe
572 the highest degree of derangement. That said, only Vitalife (see Table A) was able to normalise the
573 blood gas variables and return them to pre-diarrhoea (normal) values at 6 hours post-treatment.
574 Further assessment is required with neonatal calf diarrhoea of greater severity, however is must be
575 noted that Vitalife for Calves (see Table A) has been documented (Independent Study 1 ) as
576 correcting derangements as low as pH 6.7 within 24 hours.
577
578 INDEPENDENT STUDY 3 - CASE REPORT
579
580 Case report - Use of Vitalife for Calves® (see Table A) for the treatment of a severe case of
581 neonatal calf diarrhoea
582
583 Objective
584
585 The objective of the visit was to manage the calf diarrhoea episode on a commercial dairy farm. This
586 report focuses on one calf which, clinically, was regarded as the most severely affected calf with
587 diarrhoea.
588
589 Background
590
591 A dairy farmer contacted the veterinary herd health management team regarding a severe outbreak of
592 neonatal calf diarrhoea. The farmer reported 20 calves currently with an episode of diarrhoea, with a
593 further 10 calves dying of neonatal calf diarrhoea within the previous 5 days. The calves in this herd 594 were home-reared, and were managed in groups of 10 from birth, on deep straw bedding. Calves
595 were fed milk replacer (5 L) using manual multi-calf feeding buckets.
596
597 Case animal
598
599 A female Holstein-Friesian calf (ID: 4931 ), 15 days old. Reported to have commenced with diarrhoea
600 the previous 36 hours.
601
602 Pre-treatment clinical examination
603
604 The animal, on clinical examination, was depressed and unresponsive to stimulus. Ear positioning
605 was limp. There was no evidence of a suckle reflex and no desire to feed. Eyes were severely
606 sunken, with a corresponding estimation of dehydration of 8%. The animal stood with assistance but
607 was unable to coordinate movement. Heart auscultation revealed bradycardia with a slight murmur
608 on the left side. The animal was clinically moribund, and was given a CAS score of 4. A blood
609 sample was obtained for blood gas analysis (see Table 5 for results). This blood gas picture, with
610 particular reference to the pH, was in extremis, and amongst the lowest ever recorded in the scientific
61 1 literature.
612
613 Veterinary Diagnosis
614
615 On the basis of the blood gas and clinical assessment, the animal was regarded as being severely
616 dehydrated with a metabolic acidosis, hyponatraemia, hyperkaliaemia and hypochloridaemia.
617
618 Treatment protocol
619
620 The calf was administered Vitalife for Calves (see Table A), administered by naso-gastric tube. No
621 additional/ancillary treatments were administered.
622
623 Result
624
625 The animal was re-examined 18 hours later. Clinical assessment indicated a bright alert and
626 responsive animal, with desire to feed and a good suckle reflex. Eyes were slightly sunken and ears
627 slightly droopy. There was a willingness to walk with encouragement. The animal was given a CAS
628 score of 1. The blood gas analysis (Table 5) indicated a recovery pattern. The animal was re-
629 assessed three hours later (T+21 h) and was given a CAS score of 1. Blood gas data for this
630 timepoint is presented in Table 5 and indicated a near-normalisation of blood gas variables.
631
632 633 Conclusion
634
635 There was a marked improvement in clinical signs and evidence to support normalisation of blood gas
636 variables within 21 hours. Long-term - follow up on this animal revealed the heifer has recently
637 delivered a healthy calf.
638
639 Table 1. Description of calf husbandry regimes on each study farm until for calves in the first month
640 of life.
641
Farm Predominant Housing Shared Ad lib Milk feeding Creep calf breeds airspace water system feed with available available adult
cows
A HF Individual calf pen Yes Yes Manual multi-calf Yes, from JeX followed by groups feeding buckets 1 week of pens (up to 12 with an allowance age animals) at 3 days of 6 litres milk
of age. Deep straw replacer or whole
bedding in all pens. milk per calf per
day
B HF Individual calf pen No Yes Automatic feeders Yes, from followed by groups with an allowance 1 week of pens (up to 25 of 6 litres milk age animals) at 3 days replacer per calf
of age. Deep straw per day as a
bedding in all pens. routine. Isolated
and switched to
manual feeding if
diarrheic.
C HF Deep straw bedded No Yes Manual multi-calf Yes, from
JeX group pens from feeding buckets 1 week of birth (up to 20 with an allowance age animals) of 6 litres milk
replacer per calf
per day
D HF Straw bedded No Yes Manual multi-calf No
JeX groups pens from feeding buckets
birth (up to 10 with an allowance
animals), moving to of 4 litres milk
wood chip bedded replacer per calf
groups pens (up to per day
20 animals) from
approximately 2
weeks of age.
HF: Holstein-Friesian
JeX: Jersey cross 644 Table 2. Mean blood gas values for healthy calves and diarrhoeic calves (pre- and post- administration of ORBS - "Vitalife" - see Table A).
Blood gas variable Healthy Calves in a Diarrhoea Calves
diarrhoea-free environment Pre-treatment [range] (SEM) Post treatment [range] (SEM) (Farm A) Value (SEM) (n=28; ("=31 ) ("=31 )
72 measurements)
pH 7.42 (0.004) 7.26 [6.76, 7.39] (0.019) 7.42 [7.28, 7.49] (0.007) **
HC0 3 (mM - anion) 29.78 (0.31 1 ) 20.3 [6.1 , 27.1 ] (0.793) 30.9 [16.3, 38.9] (0.763) **
Base Excess (mM) a 6.00 (0.339) -4.9 [-31.6, 3.3] (1.173) 7.3 [-10.3, 18.6] (0.857) ** Standard
Anion Gap (mM) a 12.77 (0.405) 16.3 [9.2, 31.8] (0.865) 12.2 [5.1 , 23.7] (0.583) *
Na + (mM) 138.94 (0.317) 135.0 [1 18.4, 158.5] (1.399) 143.1 [131.4, 186.2] (1.662) **
K + (mM) 4.85 (0.040) 4.89 [3.39, 6.59] (0.122) 4.32 [3.26, 5.51] (0.018) *
CI " (mM) 99.56 (0.425) 101 .3 [84, 120] (1.265) 101 .9 [91.0, 134.0] (1.506)
SID b (mM) 44.23 (0.41 1 ) 38.59 [26.84, 52.18] (0.819) 45.53[35.43, 56.92] (0.658) **
Glucose (mM) 7.91 (0.359) 5.2 [2.5, 8.3] (0.178) 5.5 [3.7, 8.8] (0.180)
Ca + (mM) 1.26 (0.007) 1.27 [1.14, 1.42] (0.012) 1.20 [1 .01 , 1.33] ** (0.012)
Total Hb (g/dL) 1 1.67 (0.183) 13.5 [7.7, 20.0] (0.438) 12.1 [9.6, 15.7] (0.276) *
645 Statistical difference between pre- and post-treatment values estimated using linear regression, * P=0.001 , **P<0.0001
646 Calculated using blood gas machine algorithm; b SID = [Na+] + [K+] - [CI-]
Table 3. Mean blood gas values for healthy calves in a diarrhoea and diarrhoea-free environment.
Blood gas variable Healthy Calf Reference cde ranges
Diarrhea environment
Diarrhea-free environment
(Farms B, C &D) Value
(Farm A) Value (SEM)
(SEM) (n=23; 25
(n=28; 71 measurements)
measurements)
pH
7.42 (0.004) 7.39 (0.006)* 7.31 -7.53 c
HC0 3 (mM - anion)
29.78 (0.311) 30.19 (0.465) 17-29 c
Base Excess (mM) a
Standard 6.00 (0.339) 7.09 (0.502) 0±2 e
Anion Gap (mM) a
12.77 (0.405) 10.27 (0.429)* 14-20 c
Na + (mM)
138.94 (0.317) 135.88 (0.617)* 132- 152°
K + (mM)
4.85 (0.040) 4.73 (0.096) 3.9-5.8 c
CI " (mM)
99.56 (0.425) 96.64 (0.553)* 97 - 111 c
SID b (mM)
44.23 (0.411) 43.96 (0.457) 38 - 42 c
Glucose (mM)
7.91 (0.359) 5.71 (0.185)* 2.49-4.16°
Ca + (mM)
1.26 (0.007) 1.24 (0.011) > 1.0 d
Total Hb (g/dL)
11.67 (0.183) - 8.6- 11.9 d
Statistical difference between pre- and post-treatment values estimated using linear regression, * P=0.001
Calculated using blood gas machine algorithm b SID = [Na+] + [K+] - [CI-]
cAdult ranges from Smith (2015) d Adult ranges from Divers and Peek (2007) e Adult ranges from Stampfli et al. (2012).
Table 4. Spearman correlation coefficients between blood gas variables (reclassified as ordinal data) and calf clinical assessment score for all diarrhoeic study calves.
Blood gas variable Spearman correlation coefficient (rho)
PH -0.63*
HC0 3 <as anion) -0.75*
Base Excess Standard -0.74*
Anion Gap 0.40*
Na + -0.39*
K + 0.1 1
CI " -0.03
SID a -0.59*
Glucose -0.30*
Ca + -0.12
Total Hb 0.23* *P<0.05
aSID = [Na + ] + [K + ] - [Cf]
Table 5. Mean blood gas values for healthy calves and one diarrhoeic case calf (pre- and post- administration of Vitalife for Calves - see Table A).
Blood gas Normal values (SEM) Case Calf (4931 )
(n=28; 72
variable
Post treatment (T+18h, Post measurements^ P re- treatment followed by sachet 2) treatment
(TO, (T+21 h) followed by
sachet 1 )
pH 7.42 (0.004) 6.76 7.28 7.39
HC0 3 29.78 (0.31 1 ) 6.1 16.3 31.8
(mM as
anion)
Base 6.00 (0.339) -31.6 -10.3 8.5
Excess
(mM) a
Anion 12.77 (0.405) 22.8 23.7 8.7
Gap
(mM) a
Na + (mM) 138.94 (0.317) 135.3 156 131 .4
K + (mM) 4.85 (0.040) 6.54 4.01 3.26
CI " (mM) 99.56 (0.425) 1 15 121 91
SID b (mM) 44.23 (0.41 1 ) 26.84 39.01 43.66
Glucose 7.91 (0.359) 5.7 6.9 4.0 (mM)
Ca + (mM) 1 .26 (0.007) 1 .31 1 .02 1 .12
Total Hb 1 1.67 (0.183) 19.4 15.4 10.9 (g/dL)
Calculated using blood gas machine algorithm; SID
c Adopted from Independent study 1.
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