Pulsatile secretion pattern of growth hormone in dogs with pituitary-dependent hyperadrenocorticism

The amplitude and frequency of growth hormone (GH) secretory pulses are influenced by a variety of hormonal signals, among which glucocorticoids play an important role. The aim of this study was to investigate the pulsatile secretion pattern of GH in dogs in which the endogenous secretion of glucocorticoids is persistently elevated, i.e. in dogs with pituitary-dependent hyperadrenocorticism (PDH). Blood samples for the determination of the pulsatile secretion pattern of GH were collected at 10-min interval between 08:00 and 14:00 h in 16 dogs with PDH and in 6 healthy control dogs of comparable age. The pulsatile secretion patterns of GH were analyzed using the Pulsar program. GH was secreted in a pulsatile fashion in both dogs with PDH and control dogs. There was no statistical difference between the mean (+/-S.E.M.) basal GH level in dogs with PDH (0.7+/-0.1 microg/l) and the control dogs (0.6+/-0.1 microg/l). The mean area under the curve (AUC) for GH above the zero-level in dogs with PDH (4.6+/-0.6 microg/l per 6 h) was significantly lower than that in the control dogs (7.3+/-1.0 microg/l per 6 h). Likewise, the mean AUC for GH above the base-level in dogs with PDH (0.6+/-0.1 microg/l per 6 h) was significantly lower than that in the control dogs (3.7+/-1.0 microg/l per 6 h). The median GH pulse frequency in the dogs with PDH (2 pulses/6 h, range 0-7 pulses/6 h) was significantly lower (P = 0.04) than that (5 pulses/6 h, range 3-9 pulses/6 h) in the control group. The results of this study demonstrate that PDH in dogs is associated with less GH secreted in pulses than in control dogs, whereas the basal plasma GH concentrations were similarly low in both groups. It is discussed that the impaired pulsatile GH secretion in dogs with PDH is the result of alterations in function of pituitary somatotrophs and changes in supra-pituitary regulation.     

Vasopressin secretion in response to osmotic stimulation and effects of desmopressin on urinary concentrating capacity in dogs with pyometra

OBJECTIVE: To determine vasopressin (VP) secretory capacity during osmotic stimulation and the response to desmopressin treatment in dogs with pyometra and control dogs. ANIMALS: 6 dogs with pyometra before and after ovariohysterectomy and 6 control dogs. PROCEDURE: Urine osmolality (Uosm) was measured during 12 hours. Values measured on the first day defined the basal Uosm pattern. On the second day, dogs were given desmopressin to induce a desmopressin-stimulated Uosm pattern. On day 3, the VP response to osmotic stimulation was examined. RESULTS: Median Uosm on day 1 was 340 mOsm/kg (range, 104 to 1,273 mOsm/kg) and 807 mOsm/kg (range, 362 to 1,688 mOsm/kg) in dogs with pyometra before and after surgery, respectively, and 1,511 mOsm/kg (range, 830 to 1,674 mOsm/kg) in control dogs. Median Uosm during desmopressin treatment was 431 mOsm/kg (range, 168 to 1,491 mOsm/kg) and 1,051 mOsm/kg (range, 489 to 1,051 mOsm/kg) in dogs with pyometra before and after surgery, respectively, and 1,563 mOsm/kg (range, 1,390 to 2,351) in control dogs. In dogs with pyometra, threshold for VP secretion was lower before surgery (median, 340 mOsm/kg; range, 331 to 366 mOsm/kg) than after surgery (median, 358 mOsm/kg; range, 343 to 439 mOsm/kg) or in control dogs (median, 347 mOsm/kg; range, 334 to 360 mOsm/kg). Highest maximum plasma VP values were found in dogs with pyometra. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with pyometra had increased urine concentration in response to desmopressin but not to the degree of control dogs, whereas VP secretory ability was not reduced.     

Vasopressin response to osmotic stimulation in 18 young dogs with polyuria and polydipsia

Common disorders of water homeostasis leading to polyuria include a variety of endocrine, metabolic, and renal disturbances. After exclusion of most of these conditions, the diagnostic dilemma of differentiating between central diabetes insipidus, primary polydipsia, and nephrogenic diabetes insipidus may remain. Here, we report on 18 young dogs with polyuria that had been present in most cases since the dogs were puppies. The conditions were categorized according to the plasma vasopressin (VP) response to hypertonicity. The VP response to osmotic stimulation was tested by IV infusion of 20% NaCl for 2 hours. The VP response in all dogs was abnormal. Three categories could be distinguished: an exaggerated response (n = 3), a subnormal response (n = 4), and a nonlinear response with high plasma VP concentrations unrelated to increases in plasma osmolality (n = 11). The VP response to hypertonicity did not consistently distinguish among different clinical entities. In the 9 dogs with variations in urine osmolality compatible with primary polydipsia, exaggerated, subnormal, and nonlinear responses were observed. Examination of the present data questions the generally accepted notion that VP measurements during hypertonic saline infusion are the "gold standard" for the diagnostic interpretation of causes of polydipsia and polyuria. Studies of the peripheral reflection in plasma of the pulsatile VP release in healthy and polyuric individuals, with and without osmotic provocation, should be performed.     

Serum concentrations of pepsinogen A in healthy dogs after food deprivation and after feeding

OBJECTIVE: To develop and validate an ELISA for measurement of serum canine pepsinogen A (cPG A) as a diagnostic marker of gastric disorders in dogs and to measure serum cPG A in healthy dogs after food deprivation and after feeding. SAMPLE POPULATION: Sera from 72 healthy dogs. PROCEDURE: A sandwich ELISA was developed and validated. The reference range for serum concentrations of cPG A was determined in 64 healthy dogs. Postprandial changes in serum concentrations of cPG A were evaluated in 8 healthy dogs. RESULTS: Assay sensitivity was 18 microg/L, and the maximum detectable concentration was 1,080 microg/L. The observed-to-expected ratio (O:E) for 3 serial dilutions of 3 serum samples ranged from 69.3 to 104.1%. The O:E for 3 serum samples spiked with 8 concentrations of cPG A ranged from 58.8 to 120.4%. Coefficients of variation for intra- and interassay variability of 3 serum samples ranged from 7.6 to 11.9% and from 10.1 to 13.1%, respectively. Mean +/- SD serum concentration of cPG A in healthy dogs was 63.8 +/- 31.0 microg/L and the reference range was 18 to 129 microg/L. Significant increases in serum concentrations of cPG A were observed between 1 and 7 hours after feeding. CONCLUSIONS AND CLINICAL RELEVANCE: The ELISA for measuring cPG A was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use. Serum concentrations of cPG A increase substantially after feeding, which should be taken into account when conducting clinical studies.     

Pulsatile secretion of prolactin in laying and incubating turkey hens

Incubation behavior in the turkey hen is associated with a large increase in prolactin secretion. Previous research using hourly sampling of incubating hens has shown that prolactin levels fluctuate widely throughout a 24-hr period, suggestive of pulsatile secretion. This study compared the prolactin secretory patterns of laying and incubating turkeys to determine if prolactin is secreted episodically and if the high prolactin levels characteristic of the incubating hen may result, at least in part, from a change in the amplitude or frequency of secretory pulses. Blood samples were collected from cannulated, unrestrained laying and incubating hens at 10-min intervals for up to 24 hr. Data were analyzed with the PULSAR program to determine baseline prolactin levels and to establish the magnitude, frequency, and duration of episodic secretory peaks. The results revealed that prolactin is secreted in a pulsatile pattern in both laying and incubating turkey hens. Incubating hens had ninefold higher mean and baseline plasma prolactin levels than laying hens. The prolactin pulses were of approximately 12-fold greater amplitude in incubating hens than in laying hens, but the duration and frequency of pulses were the same in both groups. Therefore, the high prolactin levels required for incubation do not appear to result from an increase in the frequency of lactotroph stimulation, but rather from an increase in the prolactin secretion rate.     

Secretion pattern of thyroid-stimulating hormone in dogs during euthyroidism and hypothyroidism

In as many as one third of dogs with primary hypothyroidism a plasma thyrotropin (TSH) concentration within the reference range for euthyroid dogs is found. To determine whether this is due to fluctuations in the release of TSH, the plasma profiles of TSH were analyzed in 7 beagle bitches by collecting blood samples every 10 min for 6 hr, both before and after induction of primary hypothyroidism. After induction of primary hypothyroidism, a 37-fold increase in mean basal plasma TSH concentration and a 34-fold increase in mean area under the curve for TSH were found. Analysis by the Pulsar program demonstrated pulsatile secretion of TSH in the hypothyroid state, characterized by relatively low amplitude pulses (mean [+/-SEM]) amplitude 41 +/- 3% of basal plasma TSH level) and a mean pulse frequency of 2.0 +/- 0.5 pulses/6 hr. In the euthyroid state, significant TSH pulses were identified in only 2 dogs. The mean basal plasma TSH level correlated positively (r = 0.84) with the mean amplitude of the TSH pulses, and correlated negatively (r = -0.88) with the TSH pulse frequency. The results of this study demonstrate pulsatile secretion of TSH in dogs during hypothyroidism and only small fluctuations in plasma TSH concentrations during euthyroidism. The findings also suggest that the low TSH values occasionally found in dogs with spontaneous primary hypothyroidism may in some cases in part be the result of ultradian fluctuations.     

Effect of sevoflurane concentration on visual evoked potentials with pattern stimulation in dogs

The purpose of this study was to investigate the effects of sevoflurane concentration on canine visual evoked potentials with pattern stimulation (P-VEPs). Six clinically normal laboratory-beagle dogs were used. The minimum alveolar concentration (MAC) of sevoflurane was detected from all subjects by tail clamp method. The refractive power of the right eyes of all subjects was corrected to −2 diopters after skiascopy. For P-VEP recording, the recording and reference electrode were positioned at inion and nasion, respectively, and the earth electrode was positioned on the inner surface. To grasp the state of CNS suppression objectively, the bispectral index (BIS) value was used. The stimulus pattern size and distance for VEP recording were constant, 50.3 arc-min and 50 cm, respectively. P-VEPs and BIS values were recorded under sevoflurane in oxygen inhalational anesthesia at 0.5, 1.0, 1.5, 2.0, 2.5 and 2.75 sevoflurane MAC. For analysis of P-VEP, the P100 implicit time and N75-P100 amplitude were estimated. P-VEPs were detected at 0.5 to 1.5 MAC in all dogs, and disappeared at 2.0 MAC in four dogs and at 2.5 and 2.75 MAC in one dog each. The BIS value decreased with increasing sevoflurane MAC, and burst suppression began to appear from 1.5 MAC. There was no significant change in P100 implicit time and N75-P100 amplitude with any concentration of sevoflurane. At concentrations around 1.5 MAC, which are used routinely to immobilize dogs, sevoflurane showed no effect on P-VEP.     

Duration of nonresponse to noxious stimulation after intramuscular administration of butorphanol, medetomidine, or a butorphanol-medetomidine combination during isoflurane administration in dogs

OBJECTIVE: To assess duration of actions of butorphanol, medetomidine, and a butorphanol-medetomidine combination in dogs given subanesthetic doses of isoflurane (ISO). ANIMALS: 6 healthy dogs. PROCEDURE: Minimum alveolar concentration (MAC) values for ISO were determined. for each dog. Subsequently, 4 treatments were administered to each dog (saline [0.9% NaCl] solution, butorphanol [0.2 mg/kg of body weight], medetomidine [5.0 microg/kg], and a combination of butorphanol [0.2 mg/kg] and medetomidine [5.0 microg/kg]). All treatments were administered IM to dogs concurrent with isoflurane; treatment order was determined, using a randomized crossover design. Treatments were given at 7-day intervals. After mask induction with ISO and instrumentation with a rectal temperature probe, end-tidal CO2 and anesthetic gas concentrations were analyzed. End-tidal ISO concentration was reduced to 90% MAC for each dog. A tail clamp was applied 15 minutes later. After a positive response, 1 of the treatments was administered. Response to application of the tail clamp was assessed at 15-minute intervals until a positive response again was detected. RESULTS: Duration of nonresponse after administration of saline solution, butorphanol, medetomidine, and butorphanol-medetomidine (mean +/- SD) was 0.0+/-0.0, 1.5+/-1.5, 2.63+/-0.49, and 5.58+/-2.28 hours, respectively. Medetomidine effects were evident significantly longer than those for saline solution, whereas effects for butorphanol-medetomidine were evident significantly longer than for each agent administered alone. CONCLUSION AND CLINICAL RELEVANCE: During ISO-induced anesthesia, administration of medetomidine, but not butorphanol, provides longer and more consistent analgesia than does saline solution, and the combination of butorphanol-medetomidine appears superior to the use of medetomidine or butorphanol alone.     

Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs

Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.     

Effects of cooling dry cows under heat load conditions on mammary gland enzymatic activity, intake of food and water, and performance during the dry period and after parturition

Effect of evaporative cooling of pregnant dairy cows under heat load conditions during the dry and close-up period, on mammary gland enzymatic activity and intake of food and water, BCS, and milk performance after calving were measured in two consequent experiments. In experiment 1, 18 dry cows held in tie-stalls in a closed aerated barn under heat load conditions were used to measure the effect of evaporative cooling on the respiratory rate and body temperature, individual intake of food and water, enzymes expression level in mammary gland and adipose tissues, and BCS changes until calving. In experiment 2, two groups of 36 dry cows each, held in a commercial loose housing barn, were used to measure the effects of evaporative cooling under heat load conditions on calves' birth weight, colostrum quality and quantity, BCS changes and milk yield during 90 DIM. The non cooled (NC) cows responded to heat load by increasing their respiratory rate and daily water intake, while elevating their rectal temperature by 0.2–0.3 °C as compared with the cooled (C) cows. The external relief of heat load by the C cows in both experiments was expressed in increasing their voluntary DMI during the dry period as compared with the NC group. In experiment 2 the calves' birth weight of C cows was higher, and their colostrum quality and quantity were improved as compared with the NC group. Cooling also improved significantly BCS gain during the last 21 days until parturition, accompanied with higher cell proliferation process (based on enzymes expression at mRNA level) in the mammary gland of the C cows. Consequently, a significant increase in milk production by 5.3%, protein yield by 5.1%, ECM yield by 4.2% and FCM yield by 4.5%, was demonstrated in the C cows during 90 DIM as compared with the NC group.     

Gender, age and breed pattern of diagnoses for veterinary care in insured dogs in Sweden during 1996

More than 200,000 dogs insured for veterinary care by one Swedish company at the beginning of 1996 were included in a retrospective cross-sectional study. Data from receipts for visits to veterinarians that were part of reimbursed veterinary care events, whose cost exceeded the deductible cost, were used to calculate the risk of cause-specific morbidity, both in total and stratified by gender, age and breed. Diagnoses were described by organic system, for example, cardiovascular, and by disease process, for example, neoplastic The most commonly affected system was the integument followed by the gastrointestinal and genital systems. The genital system was the most commonly affected system in females, within which 50 per cent of the affected bitches had pyometra. Inflammatory processes were most common, followed by symptomatic conditions, traumatic injuries and neoplastic conditions.     

Arginine vasopressin stimulation of prostaglandin F2 alpha release in heifers during the estrous cycle

The effect of arginine vasopressin on the stimulation of prostaglandin F2 alpha (PGF2 alpha) release has been examined in vivo. Fifty-eight heifers received one intravenous injection of 10 IU arginine vasopressin on either Day 0 (n = 14), Day 6 (n = 12), Day 13 (n = 14) and Day 18 or 19 or 20 (Day 18-20, n = 18) after the onset of oestrus (Day 0) to determine the effect of arginine vasopressin at different times of the oestrous cycle. Frequent blood samples were taken before and after arginine vasopressin injection for the measurement of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) by radioimmunoassay (RIA). Blood samples for progesterone determinations were taken 2 hr before and 24 hr after arginine vasopressin to monitor luteal function. The data show that arginine vasopressin causes an increase (P less than 0.005) in PGFM concentrations only at Day 18-20 of the cycle in 67% of the experimental heifers.     

Hematological, osmotic, and scanning electron microscopic study of erythrocytes of dogs given beta-acetylphenylhydrazine

Hematologic examinations, osmotic fragility tests, and scanning electron microscopy of erythrocytes were done on blood of dogs given 5 mg/kg of beta-acetylphenylhydrazine for 5 weeks. Reticulocytes, Heinz bodies, and serum total bilirubin values increased in the 1st week. Reticulocyte numbers peaked in the 2nd week, and reticulocytosis persisted through the 5th week. Erythrocyte, packed cell volume, and hemoglobin values decreased markedly and became lowest in the 2nd week. Mean corpuscular volume increased in the 1st week and remained increased for the duration of treatment. Erythrocyte osmotic fragility was increased after 1 week of treatment. Echinocytes were increased with a peak level of 47.6% at week 1 of treatment. Increased numbers of acanthocytes and schizocytes also were detected.     

Micronized fibres affect in vitro fermentation under normal buffered and osmotic stress conditions using porcine inocula

In this in vitro study, the modified Hohenheim gas test was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting a standard pig diet with varying levels of crude protein (CP; 20, 24 and 28% CP), and supplemented with one of three fibre sources manufactured by micronization treatment. These were wheat envelopes (MWE), pea fibre (MPF) and lupine fibre (MLF). For comparison, inulin was used. As intestinal bacteria have to cope with varying osmotic conditions in their ecosystem, fermentation was performed under normal buffered and osmotic stress conditions. After 24 h of fermentation, total gas production and ammonia production were measured. In addition, the effect of MWE and inulin on short‐chain fatty acid (SCFA) production and numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp., Enterobacteriaceae, Enterococcus spp., Clostridium cluster XIVa and Clostridium cluster IV, were determined using quantitative real‐time PCR. Under normal buffered conditions, supplementation of MWE resulted in increased (p < 0.05) SCFA, acetic, propionic and valerianic acid production at CP levels of 20 and 28%. There was an increase (p < 0.05) in ammonia production for the micronized supplements, and for MWE an increased (p < 0.05) branched‐chain proportion was observed, possibly due to higher availability of protein for fermentation which was released during the micronization process. Osmotic stress conditions reduced (p < 0.05) total gas as well as total SCFA, acetic and propionic acid production for all treatments, while cell counts were increased (p < 0.05) for Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. Under normal buffered conditions in combination with 24 and 28% CP levels, lactobacilli were increased for MWE, compared to inulin (p < 0.05). In conclusion, micronized supplements such as MWE may beneficially modulate pigs' intestinal microbiota by increasing SCFA production in addition to a selective proliferation of lactobacilli.     

Cardiovascular and respiratory effects of ketamine infusions in isoflurane-anesthetized dogs before and during noxious stimulation

OBJECTIVE: To characterize the effects of ketamine administration on the cardiovascular and respiratory systems and on acid-base balance and to record adverse effects of ketamine in isoflurane-anesthetized dogs. ANIMALS: 6 healthy adult mongrel dogs. PROCEDURE: Dogs were anesthetized with isoflurane (1.25 times the individual minimum alveolar concentration) in oxygen, and ketamine was administered IV to target pseudo-steady-state plasma concentrations of 0, 0.5, 1, 2, 5, 8, and 11 microg/mL. Isoflurane concentration was reduced to an equipotent concentration. Cardiovascular, respiratory, and acid-base variables; body temperature; urine production; and adverse effects were recorded before and during noxious stimulation. Cardiac index, stroke index, rate-pressure product, systemic vascular resistance index, pulmonary vascular resistance index, left ventricular stroke work index, right ventricular stroke work index, arterial oxygen concentration, mixed-venous oxygen concentration, oxygen delivery, oxygen consumption, oxygen extraction ratio, alveolar-arterial oxygen partial pressure gradient, and venous admixture were calculated. Plasma ketamine and norketamine concentrations were measured. RESULTS: Overall, ketamine administration improved ventilation, oxygenation, hemodynamics, and oxygen delivery in isoflurane-anesthetized dogs in a dosedependent manner. With the addition of ketamine, core body temperature was maintained or increased and urine production was maintained at an acceptable amount. However, at the higher plasma ketamine concentrations, adverse effects such as spontaneous movement and profuse salivation were observed. Myoclonus and dysphoria were observed during recovery in most dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of ketamine appears to be a suitable technique for balanced anesthesia with isoflurane in dogs. Plasma ketamine concentrations between 2 to 3 microg/mL elicited the most benefits with minimal adverse effects.     

Fluid and electrolyte shifts during and after Agility competitions in dogs

This research assesses the relative contribution of splenic contraction and fluid shifts out of the vascular compartment to the increases in packed cell volume associated with Agility exercises. It also aims to evaluate the changes in the concentrations of electrolytes and markers of hydration state. Fifteen dogs of both sexes were subjected to an Agility exercise of an approximate duration of 100 s. Blood samples were obtained within the first 30 s after competition and at 5, 15, and 30 min of recuperation. Resting values were established previously. The following parameters were determined: packed cell volume (PCV), plasma lactate (LA), total plasma protein (TPP), albumin (ALB), urea (BUN), creatinine (CREA), chloride (Cl), calcium (Ca), phosphorus (P), sodium (Na) and potassium (K). Changes in plasma volume (PV), total RBC volume (V(RBC)) and blood volume (BV) were calculated immediately after exercise and at 30 minutes of recovery. It was found that during Agility competition, BV, V(RBC) and PV increased 12, 21 and 4% respectively, indicating that the spleen contraction was the main determinant on the increase of BV. In comparison with resting values, BV decreased after recuperation (-5%), due to the recapture of erythrocytes by the splenic reserve (V(RBC), -12%). Additionally, Agility exercise induced increases in plasma Cl and LA, with significant reductions of ALB, Ca and P and absence of modifications in Na, K, BUN and CREA concentrations.     

Detection of biofilm formation and nanobacteria under long-term cell culture conditions in serum samples of cattle,goats, cats,and dogs

OBJECTIVE: To determine the prevalence of biofilm formation under long-term cell culture conditions in serum samples of dairy cattle, goats, cats, and dogs, and to determine whether there is an association between nanobacteria and biofilm formation. SAMPLE POPULATION: Serum samples of clinically normal animals (313 dairy cattle, 48 goats, 140 dogs, and 44 cats) and animals with various medical conditions (60 dogs and 116 cats). PROCEDURE: Serum was incubated under cell culture conditions and observed for biofilm formation by use of light microscopy, electron microscopy, and spectroscopy. A polymerase chain reaction assay was developed to identify 16S rRNA gene sequences of nanobacteria. RESULTS: Biofilm formation developed in serum samples of 304 of 313 (97%) cattle, 44 of 48 (92%) goats, 44 of 44 (100%) cats, and 126 of 140 (90%) dogs. Prevalence of serum samples with positive results for biofilm formation was not significantly different between cats or dogs with and without medical conditions associated with pathologic extraskeletal calcification processes. Scanning electron microscopy and spectroscopy of biofilm samples revealed small coccoid particles consisting mainly of calcium and phosphate. Polymerase chain reaction assay failed to amplify sequences of nanobacteria. CONCLUSIONS AND CLINICAL RELEVANCE: Under long-term cell culture conditions, biofilm made up of aggregates of calcium and phosphate crystals does form in serum samples of clinically normal dairy cattle, goats, cats, and dogs. Disease, however, does not predispose to biofilm formation in serum samples of dogs and cats. Our findings did not support the existence of nanobacteria in serum samples of cattle, goats, cats, and dogs.